Posterior eye diseases are currently treated by intravitreal injection and sometimes by systemic drug administration. Systemic administration is not preferred because of the systemic toxicity encountered. Intravitreal injection is a more effective route of administration compared with systemic drug delivery, but repeated injections can cause severe side effects to the eye and involve high healthcare cost. There is an unmet need of a more effective drug delivery method in the treatment of posterior eye diseases. RNA nanotechnology provides molecules that has the simplicity in design with the characteristics of DNA and can be used in therapies. However, a major problem in RNA nanotechnology is that RNA molecules are relatively unstable, e.g., RNA degradation in vivo and dissociation at ultra-low concentration after administration into the body. Recently, RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor have been found to be thermodynamically and chemically stable both in vitro and in vivo. These pRNA nanoparticles, with their scaffolds purely made up of RNA, can be modified to harbor multiple modules with different functionalities such as RNA aptamer, reporter moiety, and therapeutic siRNA, miRNA, or other chemical drugs or ligands as subunits all in the same single nanoparticle. Our preliminary study on the distribution of pRNA nanoparticles in the eye has shown that one of these pRNA nanoparticles are internalized in the cells in the retina after subconjunctival injection in vivo. This suggests the potential of pRNA nanoparticles via subconjunctival injection as an efficient drug delivery system of RNA-based therapeutic agents to the cells in the retina for treating posterior eye diseases. However, the nanoparticle was cleared from the subconjunctival pocket quickly after the injection. The objectives of the present project are to (a) evaluate ocular delivery of pRNA nanoparticles with different module subunits via subconjunctival injection and (b) develop a sustained delivery system for these nanoparticles. A temperature sensitive gel system, which is a liquid at room temperature and solidifies at body temperature after subconjunctival injection, will be used as the sustained delivery system to enhance the retention of pRNA in the subconjunctival space. In this project, ocular delivery of pRNA nanoparticles will first be evaluated. The mechanisms of pRNA delivery to the retina after subconjunctival injection will be investigated. To overcome fast clearance after subconjunctival injection, a temperature sensitive gel system will be developed to provide sustained ocular delivery of the pRNA nanoparticles. The ultimate goal of this project is to develop a drug delivery platform for ocular drug delivery utilizing pRNA nanoparticles to provide an effective and less invasive approach than intravitreal injection.